Centre for Regenerative Medicine and Devices, University of Brighton, Huxley Building Lewes Road, Brighton, BN2 4GJ, UK.
School of Applied Sciences, University of Brighton, Huxley Building Lewes Road, Brighton, BN2 4GJ, UK.
J Mater Sci Mater Med. 2024 Mar 25;35(1):19. doi: 10.1007/s10856-024-06785-z.
The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads' size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells' ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.
干细胞疗法的疗效取决于移植细胞逃避早期免疫反应的能力和在移植部位的保留能力。已经提出使用组织工程支架或可注射生物材料作为载体，但它们仍然存在与可靠的制造工艺、手术实践和临床结果相关的局限性。藻酸盐微球是封装间充质基质细胞的潜在候选物，目的是提供适合微创和无支架移植、组织粘附特性和免受免疫反应的输送载体。然而，稳定微球的形成依赖于藻酸盐与二价钙离子的交联，而这种交联浓度对细胞是有毒的，这使得对微球大小和单细胞包封的控制不可靠。本工作展示了一种创新的、高通量的、可重复的微流控系统生产单细胞、无钙藻酸盐包被的人间充质基质细胞的效率。在测试的各种条件下，通过 DAPI 染色细胞核后的可见光和共聚焦显微镜显示，微流控系统以 2% w/v 的藻酸盐微胶囊以 2000 个细胞/分钟的速度产生最佳的单细胞包封，这些微胶囊具有可重复的形态和平均尺寸为 28.2 ± 3.7 µm。通过微球相对快速地黏附在组织培养塑料上以及细胞在 24 小时后逐渐破坏微胶囊壳并增殖的能力，证明了藻酸盐微胶囊的粘附特性、包封细胞的活力及其逃脱藻酸盐微胶囊的能力。为了模拟移植后早期的炎症反应，将包封的细胞暴露于增殖的巨噬细胞中，细胞接种密度不同，持续时间长达 2 天，并通过延时显微镜评估微胶囊对细胞的保护作用，结果显示微胶囊的保护作用长达 48 小时。这项工作强调了微流控系统通过良好的制造实践标准精确包封细胞的潜力，同时有利于细胞在基质上的保留、活力和增殖。
